Cannabinoid receptors mediate the psychotropic effects of marijuana, as well as those of therapeutic potential, including antinociceptive and immunomodulatory responses. Acylethanolamide (AEA) and 2-monoacyl glycerol (2-MAG) are endogenous cannabinomimetics, and are simply the nonphosphorylated counterparts of the LPA receptor agonists N-acyl ethyl phosphoric acid (NAEPA) and sn-2 LPA, respectively. Further, the structural similarity between the two classes of agonists suggests that sn-2 LPA and NAEPA may be cannabinoid precursors. The characterization of membrane bound phosphatidic acid phosphatases (PAP-2) suggests that these enzymes may provide the enzymatic link between the two classes of lipids. This proposal is designed to test the hypothesis that the biosynthesis of the cannabinomimetics 2-MAG and AEA occurs via PAP-2 mediated hydrolysis of LPA and NAEPA, and the latter is in turn the product of phospholipase C cleavage of N-acyl phosphatidylethanolamine (NAPE). First, the pharmacological and biochemical characterization of PAP-2 isoforms will be extended by determining if sn-2 LPA and NAEPA with long chain, polyunsaturated fatty acyl moities are PAP-2 substrates. Additionally, the predicted extracellular orientation of the PAP-2 active site will be tested experimentally, and structural analogs of PAP-2 substrates will be screened as blockers of PAP-2 activity. Secondly, mass spectroscopy will be employed to determine if NAEPA is detectable in rat brain and if it is present at sufficiently high quantities to support the proposed role as LPA receptor agonist and AEA precursor. Next, neuronal cell lines homogenates will be assayed for the presence of a PLC activity using radiolabelled NAPE as substrate. Finally, to examine whether the generation of cannabinoid ligands by PAP-2s may be physiologically relevant, in situ hybridization will be used to determine if the phosphatases are expressed in the same brain regions as cannabinoid receptors. Herein, a novel biosynthetic route for endogenous cannabinoid ligands is proposed, which may be functionally coupled to a second family of bioactive lipids, the LPA receptor agonists.